Genomic imprinting is the unequal expression of the maternal and paternal alleles of a gene. Insulin like growth factor II (Igf2) and its neighboring gene, H19, are subject to imprinting, with Igf2 being expressed only from the paternal allele and H19, only the maternal. Normal expression of Igf2 is necessary for proper regulation of fetal body size and is likely to be an important variable in human birth weight. Abnormal imprinting of Igf2 is one cause of the genetic disease Beckwith-Wiedemann syndrome, which is characterized by fetal overgrowth and abdominal wall defects. In cancer, overexpression of Igf2 is common and is thought to increase the size and number of tumors. The normal imprinted expression of these two genes depends on a methylation sensitive chromatin boundary known as the ICR. Chromatin boundaries are DNA elements that isolate genes from surrounding influences by preventing the encroachment of repressive heterochromatin and by blocking the activity of enhancers of neighboring genes. Using a twist in enhancer blocking activity, the unmethylated maternal ICR inhibits Igf2 expression by preventing its own enhancers from activating the maternal copy. The paternal ICR, however, is methylated and has no boundary activity, which allows the enhancers to activate paternal Igf2. The maternal ICR's blocking activity requires the binding of CTCF, a zinc finger transcription factor found at all known vertebrates boundaries. On the paternal ICR, DNA methylation acts as switch by directly inhibiting CTCF binding to its recognition site. The long term objectives of this proposal are to understand how the ICR and genomic imprinting regulate the expression of Igf2 and H19. Specifically, this proposal addresses how CTCF accomplishes enhancer blocking, what other proteins cooperate with CTCF, and what DNA sequences are required for paternal ICR methylation. Enhancer blocking has been implicated in the regulation of many genes and is likely to be a fundamental form of gene regulation. Similarly, understanding ICR allele specific methylation will apply not only to other imprinted genes, but will be applicable to methylation of biallelic genes in development and disease.